Method for detecting VISTA protein in tumor cell line through immunoblotting method

Abstract

The invention discloses a method for detecting VISTA protein in a tumor cell line by an immunoblotting method. The method comprises a step of removing glycosylation modification of VISTA. Especially for colorectal cancer tumor cell lines, lung cancer tumor cell lines or prostatic cancer tumor cell lines. After the glycosylation modification of the VISTA is removed, it can be obviously detected that the protein of the VISTA is converted into a highlight band with the size of about 36 kD from 40-70 kD towing, and the detection efficiency of Western blots is improved. Through the deglycosylation treatment, the expression of VISTA in colorectal cancer cells can be more effectively detected, and the method is very critical to accurate identification of patient groups which may benefit from VISTA monoclonal antibody treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical detection technology, and in particular relates to a method for detecting VISTA protein in a tumor cell line by immunoblotting. Background Art

[0002] VISTA (V-domain immunoglobulin suppressor of T cell activation) is a newly discovered immunosuppressive checkpoint molecule in recent years. It is similar in structure to PD-L1 and belongs to the B7 family of immune checkpoints. VISTA can be expressed in T lymphocytes (including CD4+ and CD8+ T lymphocytes), and is significantly highly expressed in myeloid monocytes / macrophages, neutrophils and dendritic cells, and can also be seen in some tumor cells. VISTA mainly inhibits T cell activation, thereby allowing tumor cells to escape the body's immune recognition and attack. Specific blocking of VISTA can significantly enhance the anti-tumor immune response. However, the traditional Western Blot method (Western Blots) has poor detection effect on VISTA in colorectal cancer cell lines, and the VISTA protein has a drag at 40-70kD. Summary of the invention

[0003] To this end, in view of the technical problem that the existing Western Blots method has a poor detection effect on VISTA in colorectal cancer cell lines, the present invention provides a method for detecting VISTA protein in tumor cell lines by Western blotting, characterized in that the method includes a step of removing the glycosylation modification of VISTA.

[0004] Among them, after the glycoprotein is denatured, a step of removing the glycosylation modification of VISTA is performed.

[0005] Preferably, the tumor cell line is a colorectal cancer tumor cell line, a lung cancer tumor cell line or a prostate cancer tumor cell line.

[0006] Preferably, the method comprises the following steps:

[0007] Step S1, extracting and lysing tumor cells in a tumor cell line to obtain cell proteins;

[0008] Step S2, denaturing the cell protein into glycoprotein;

[0009] Step S3, removing the glycosylation modification of VISTA;

[0010] Step S4, protein concentration determination and glue preparation;

[0011] Step S5, electrophoresis and membrane transfer;

[0012] Step S6, first blocking, then incubating with primary antibody and secondary antibody;

[0013] Step S7: washing and developing.

[0014] Preferably, the method comprises the following steps:

[0015] Step S1, collecting the culture dish where the tumor cell line adherent cells are located, aspirating the supernatant, washing twice with PBS to wash away the residual culture medium, adding about 200 to 500 μL of lysis solution into the culture dish, scraping the cells from the culture dish, transferring them into an EP tube, and ultrasonically lysing them to obtain a cell lysate of cell protein;

[0016] Step S2, taking the cell lysate, adding 10× glycoprotein denaturation buffer, mixing by pipetting, incubating at 100° C. for 8 to 12 minutes, and then rapidly cooling on ice;

[0017] Step S3, taking the denatured glycoprotein product, adding sodium phosphate buffer at pH 7.5, NP40 and double distilled water to prepare a reaction system, then adding N-glycosidase F, and incubating at 37° C. overnight;

[0018] Step S4, according to the instructions of the kit, add the protein supernatant obtained by centrifugation and the G250 reagent, gently blow and mix evenly, add the mixed solution to each well of the 96-well plate, and then measure the absorbance at 595nm with an ELISA reader, calculate the protein concentration according to the standard curve, and mark the tube; the gel rapid preparation kit is configured with a separation gel of corresponding concentration;

[0019] Step S5, fix the gel in the electrophoresis tank, add a sufficient amount of electrophoresis solution, add the protein sample to the corresponding electrophoresis tank, use 80V voltage in the concentrated gel, and use 120V voltage for electrophoresis when the protein and marker are separated and enter the lower separation gel; place the transfer sponge, transfer filter paper, gel, NC membrane, transfer filter paper and transfer sponge in order, clamp them with transfer clips, insert them into the transfer tank, add an ice box, fill with transfer solution, and transfer at 280mA to 320mA for 0.8 to 1.2 hours under constant current conditions;

[0020] Step S6, soaking the transferred NC membrane in 4-6% skim milk, blocking at room temperature for 0.8-1.2 hours; cutting the target band area from the NC membrane, soaking it in 4-6% skim milk containing VISTA primary antibody at a primary antibody concentration of 1:800-1200, placing it on a shaker, incubating at 4°C overnight or at room temperature for 1.5-2.5 hours; recovering the 4-6% skim milk diluted with the primary antibody, washing the NC membrane with TBST for 2-5 times, each time for 4-8 minutes, and then preparing the secondary antibody corresponding to the primary antibody with TBST, placing it on a shaker, and incubating it at room temperature for 1 hour;

[0021] Step S7, after the secondary antibody incubation is completed, the NC membrane is washed with TBST for 2 to 5 times, each time for 4 to 8 minutes; the developer is prepared in a volume ratio of 1:0.8 to 1.2, and placed in a luminometer for development.

[0022] Preferably, the method comprises the following steps:

[0023] Step S1, collecting the culture dish where the tumor cell line adherent cells are located, aspirating the supernatant, washing twice with PBS to wash away the residual culture medium, adding about 200 to 500 μL of lysis solution into the culture dish, scraping the cells from the culture dish, transferring them into an EP tube, and ultrasonically lysing them to obtain a cell lysate of cell protein;

[0024] Step S2, take 18 μL of cell lysate, add 2 μL of 10× glycoprotein denaturation buffer, mix by pipetting, incubate in a 100°C metal bath for 10 minutes, and then quickly cool on ice;

[0025] Step S3, taking 10 μL of the glycoprotein denatured product, adding 2 μL of 500 mM sodium phosphate buffer at pH 7.5, 2 μL of 10% NP40 and 6 μL of double distilled water to prepare a 20 μL reaction system, then adding 1 μL of N-glycosidase F (PNGase F), and incubating at 37° C. overnight;

[0026] Step S4, according to the Bradford kit instructions, add the protein supernatant obtained by centrifugation and G250 reagent at a volume ratio of 10:300, gently blow evenly, add 300 μL of the mixture to each well of the 96-well plate, then measure the absorbance at 595 nm with an ELISA reader, calculate the protein concentration according to the standard curve, and mark the tube; the rapid gel preparation kit is configured with a separation gel of corresponding concentration;

[0027] Step S5, fix the gel in the electrophoresis tank, add a sufficient amount of electrophoresis solution, add the protein sample to the corresponding electrophoresis tank, use 80V voltage in the concentrated gel, and use 120V voltage for electrophoresis when the protein and marker are separated and enter the lower separation gel; place the transfer sponge, transfer filter paper, gel, NC membrane, transfer filter paper and transfer sponge in order, clamp them with transfer clips, insert them into the transfer tank, add an ice box, fill them with transfer solution, and transfer at 300mA under constant current conditions for 1 hour;

[0028] Step S6, soak the transferred NC membrane in 5% skim milk and block at room temperature for 1 hour; cut the target band area from the NC membrane, soak it in 5% skim milk containing VISTA primary antibody at a primary antibody concentration of 1:1000, place it on a shaker, and incubate it at 4°C overnight or at room temperature for 2 hours; recover the 5% skim milk diluted with the primary antibody, wash the NC membrane with TBST for 3 times, 5 minutes each time, and then prepare the secondary antibody corresponding to the primary antibody with TBST, place it on a shaker, and incubate it at room temperature for 1 hour;

[0029] Step S7, after the secondary antibody incubation is completed, the NC membrane is washed 3 times with TBST, each time for 5 minutes; the developer is prepared in a 1:1 volume ratio and placed in a luminometer for development.

[0030] Preferably, the protein lysis solution in step S1 is prepared by adding 20 mg / mL PMSF, 100 mmoL sodium fluoride, 200 mmoL sodium vanadate and protease inhibitors to 1% NP40 at a volume ratio of 1:100 and shaking gently to obtain the solution.

[0031] Preferably, the glycoprotein denaturation buffer is a 0.5% SDS sodium dodecyl sulfate solution containing 40 mM dithiothreitol.

[0032] The positive progress of the present invention is that in the method of detecting VISTA protein in tumor cell lines by immunoblotting of the present invention, especially in colorectal cancer tumor cell lines, lung cancer tumor cell lines or prostate cancer tumor cell lines, after removing the glycosylation modification of VISTA, it can be clearly detected that the VISTA protein is converted from a 40-70kD drag to a bright band of about 36kD, which improves the test efficiency of Western blots. Through deglycosylation treatment, the expression of VISTA in colorectal cancer cell lines can be more effectively detected, which is extremely critical for accurately identifying patient groups that may benefit from VISTA monoclonal antibody treatment.

[0033] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A Analyze the expression of VISTA in various cell subpopulations for single-cell data;

[0035] Figure 1B Analyze the expression of VISTA in pan-cancer cell lines for the CCLE database;

[0036] Figure 1C Western blots were used to detect the protein expression of VISTA in a panel of colorectal cancer cell lines;

[0037] Figure 2A Western blots were used to detect changes in protein expression of VISTA in colorectal cancer cell lines after PNGase F treatment;

[0038] Figure 2B Western blots were used to detect changes in VISTA protein expression in lung cancer and prostate cancer cell lines after PNGase F treatment;

[0039] Figure 2C After PNGase F treatment, the expression changes of VSITA in colorectal cancer cells were detected by cytofluorescence (IF);

[0040] Figure 2D After treatment with the glycosylase inhibitor TM, the protein expression changes of VISTA in colorectal cancer cell lines were detected;

[0041] Figure 2E After treatment with glycosylase inhibitors NGI-1 and TM, the expression changes of VSITA in colorectal cancer cells were detected by cytofluorescence (IF);

[0042] Figure 3A and Figure 3B Representative images (scale bar = 20 μm) and H-score change graphs of VISTA staining intensity changes in tumor cells and stromal cells in colorectal cancer tissues treated with PNGase F deglycosylation compared with those without treatment, respectively;

[0043] Figure 3C and Figure 3D These are the trends of VISTA staining changes in tumor cells and stromal cells in colorectal cancer tissues treated with PNGase F deglycosylation compared with those without treatment;

[0044] Figure 3E This is the relationship between the difference in VISTA expression and the OS and DFS of patients after PNGase F deglycosylation treatment. DETAILED DESCRIPTION

[0045] In response to the phenomenon that "the positive detection rate of VISTA in colorectal cancer tumor cells is only 1.8-2.6%" (refer to Zong L, Yu S, Mo S, Zhou Y, Xiang Y, Lu Z, et al. High VISTA Expression Correlates With a Favorable Prognosis in Patients With Colorectal Cancer. Journal of immunotherapy (Hagerstown, Md: 1997), 2021; 1: 22-28.), the applicant first detected the expression levels of VISTA mRNA and protein in colorectal cancer tumor cells to determine whether the low IHC detection rate of VISTA in colorectal cancer tumor cells is due to its own low expression or because other reasons affect the IHC detection efficiency.

[0046] By analyzing the single-cell public database, the applicant found that VISTA was transcriptionally activated in 13.1% of colorectal cancer tumor cells ( Figure 1A ), and through pan-cancer tumor cell transcriptome analysis, it was found that VISTA is expressed in various tumor cells including colorectal cancer ( Figure 1B ). Subsequently, at the protein level, the applicant found that all colorectal cancer cell lines highly expressed VISTA, but from the Western Blots images, it can be clearly found that the VISTA protein is dragged, suggesting that VISTA has post-translational modification ( Figure 1C ). So could it be that post-translational modification of the protein affects the detection rate of VISTA in IHC? The applicant made various attempts and surprisingly found that removing the glycosylation level of VISTA can effectively increase the IHC positive rate of VISTA in colorectal cancer tumor cells. The specific embodiment process is listed as follows:

[0047] Example 1 IHC detection of VISTA in colorectal cancer tissue sections

[0048] Slice baking: Insert the colorectal cancer tissue slices into the slice rack and bake them in a 63°C oven for at least 90 minutes.

[0049] Dewaxing: Soak the baked tissue sections in the following order: xylene No. 1 (5 minutes) → xylene No. 2 (5 minutes) → anhydrous ethanol No. 1 (5 minutes) → anhydrous ethanol No. 2 (5 minutes) → 95% ethanol No. 1 (3 minutes) → 95% ethanol No. 2 (3 minutes) → 75% ethanol No. 1 (3 minutes) → 75% ethanol No. 2 (3 minutes). After the end, wash with plenty of water for 5 minutes.

[0050] Antigen repair: Place tissue sections in a histochemical box, fill with citric acid antigen repair solution, heat in a microwave oven for 10 minutes, open the microwave door, let it cool naturally for 5 minutes, heat in the thawing mode for another 10 minutes, then take out the histochemical box and place it in water to cool down. When the temperature drops to room temperature, wash with PBS 3 times, 5 minutes each time.

[0051] Glycoprotein denaturation: The tissue sections were incubated with 1× glycoprotein denaturation buffer (0.5% SDS, 40 mM DTT) at room temperature for 3 hours, and then washed 4 times with PBS for 5 minutes each time.

[0052] Deglycosylation: Tissue sections were incubated with PNGase F (NEB, #P0704L) diluted to 5% in PBS at 37°C overnight, and then washed three times with PBS for 5 minutes each time.

[0053] Inactivation: First, circle the tissue area with an immunohistochemistry brush, then cover the tissue area with 30% hydrogen peroxide diluted to 3% with methanol, inactivate peroxidase for 30 minutes in the dark, and then wash with PBS 3 times, 5 minutes each time.

[0054] Blocking: Cover the tissue with 5% goat serum for blocking at room temperature for 1 hour.

[0055] Incubate with primary antibody: shake off the goat serum used for blocking on the sections, prepare VISTA primary antibody with 5% goat serum (volume ratio of 1:100, antibody information: CST, #64953), place in a humidified box, and incubate overnight at 4°C.

[0056] Rewarming: Take the wet box containing the slices out of the 4°C refrigerator, open the lid, and let it stand at room temperature for about 30 minutes to allow it to rewarm naturally.

[0057] Incubation with secondary antibody: Wash the rewarmed sections with PBS three times, 10 minutes each time. Then drop the mouse and rabbit universal secondary antibody (Gene Technology (Shanghai) Co., Ltd.) on the tissue and incubate at room temperature for 1 hour.

[0058] DAB color development: After removing the secondary antibody, the sections were washed with PBS for 3 times, 10 minutes each time. DAB color developer (Gene Technology (Shanghai) Co., Ltd.) was prepared at a volume ratio of 1:25 and color was developed under a microscope.

[0059] Hematoxylin counterstaining: Stain the tissue area in the section covered with hematoxylin for 1 minute, then rinse with plenty of water for 20 minutes.

[0060] Transparent sealing: Soak the tissue sections in the following order: 75% ethanol No. 1 (5 minutes) → 75% ethanol No. 2 (5 minutes) → 95% ethanol No. 1 (5 minutes) → 95% ethanol No. 2 (5 minutes) → anhydrous ethanol No. 1 (3 minutes) → anhydrous ethanol No. 2 (5 minutes) → xylene No. 1 (10 minutes) → xylene No. 2 (10 minutes). Finally, seal the sections with neutral resin.

[0061] Photography: After the slides have been air-dried for 24 hours, they can be used for observation and photography under a microscope.

[0062] Example 2 Western Blots Detection of VISTA in Colorectal Cancer Cell Lines

[0063] Prepare protein lysis buffer: add PMSF (20 mg / mL), sodium fluoride (100 mmoL), sodium vanadate (200 mmoL), and protease inhibitor (PI) to 1% NP40 at a volume ratio of 1:100, and shake gently to make it uniform.

[0064] Extract cell protein: Collect the culture dish where the adherent cells are to be extracted, remove the supernatant, wash twice with PBS to remove the residual culture medium, add about 200-500 μL lysis solution to the culture dish, scrape the cells from the culture dish,

[0065] Transfer to a 1.5 mL EP tube and perform ultrasonic lysis.

[0066] Glycoprotein denaturation: Take 18 μL of cell lysate, add 2 μL of 10× glycoprotein denaturation buffer (5% SDS, 400 mM DTT), mix by pipetting, incubate in a 100°C metal bath for 10 minutes, and then quickly cool on ice.

[0067] Deglycosylation: Take 10 μL of glycoprotein denatured product, add 2 μL of 500 mM sodium phosphate buffer (pH 7.5), 2 μL of 10% NP40 and 6 μL of double distilled water to make a 20 μL reaction system, then add 1 μL of PNGase F and incubate at 37°C overnight.

[0068] Protein concentration determination: According to the Bradford kit instructions, add the protein supernatant obtained by centrifugation and G250 (detergent compatible) reagent at a volume ratio of 10:300, and gently pipette evenly. Add 300 μL of the mixture to each well of the 96-well plate, then measure the absorbance (OD value) at 595 nm with an ELISA reader, calculate the protein concentration according to the standard curve, and mark the tube.

[0069] Gel preparation: Use the rapid gel preparation kit of Shanghai Yazyme Biopharmaceutical Technology Co., Ltd. to prepare the separation gel of corresponding concentration.

[0070] Electrophoresis: Fix the gel in the electrophoresis tank, add enough electrophoresis solution, and then add the protein sample to the corresponding electrophoresis tank. Use 80V voltage in the concentrated gel, and when the protein and marker are separated and enter the lower separation gel, use 120V voltage for electrophoresis.

[0071] Transfer: Place the transfer sponge, transfer filter paper, glue, NC membrane, transfer filter paper and transfer sponge in order, clamp them with transfer clips, insert them into the transfer tank, add ice box, and fill with transfer solution. Transfer at 300mA for 1 hour under constant current conditions.

[0072] Blocking: Soak the transferred NC membrane in 5% skim milk and block it at room temperature for 1 hour.

[0073] Incubate with primary antibody: Cut the target band area from the NC membrane, soak it in 5% skim milk containing VISTA primary antibody (CST, #64953) (primary antibody concentration 1:1000), place it on a shaker, and incubate it at 4°C overnight or at room temperature for 2 hours.

[0074] Incubate with secondary antibody: Recover the 5% skim milk diluted with primary antibody, wash the NC membrane with TBST for 3 times, 5 minutes each time, then prepare the secondary antibody corresponding to the primary antibody with TBST, place on a shaker, and incubate at room temperature for 1 hour.

[0075] Washing: After the secondary antibody incubation, the NC membrane was washed three times with TBST, 5 minutes each time.

[0076] Development: Prepare Millipore ECL developer in a 1:1 volume ratio and place in a luminometer for development.

[0077] Example 3IF detection of VISTA in colorectal cancer cell lines

[0078] Slide preparation: Colorectal cancer cells were inoculated into a 24-well plate with slides placed in advance. After 2 days, the cells grew on the slides and the culture medium in the wells was aspirated and the plates were washed 3 times with PBS.

[0079] Fixation: Add 1 mL of 4% paraformaldehyde for fixation at room temperature for 15 minutes, and then wash three times with PBS.

[0080] Glycoprotein denaturation: Add 1× glycoprotein denaturation buffer (0.5% SDS, 40 mM DTT) to the wells, incubate at 100°C for 10 minutes, then quickly cool on ice, and then wash three times with PBS.

[0081] Deglycosylation treatment: The slides were incubated with 5% PNGaseF diluted with PBS at 37°C overnight, and then washed three times with PBS.

[0082] Permeabilization: Add 1 mL of 0.2% TritonX-100 and perform permeabilization at room temperature for 10 minutes, then wash three times with PBS.

[0083] Blocking: Add 1 mL of 5% goat serum for blocking at room temperature for 1 hour, and then wash three times with PBS.

[0084] Antibody incubation: VISTA primary antibody (CST, #64953) was added to 5% goat serum at a volume ratio of 100:1, incubated at room temperature for 1 hour, washed 3 times with PBS, and then added with the corresponding secondary antibody and incubated at room temperature for 1 hour, and then washed 3 times with PBS.

[0085] Sealing observation: Use DAPI to stain for 15 minutes in the dark, wash with PBS three times, and then add anti-fluorescence quencher to seal the slide. Observe the expression of VISTA in cells under a fluorescence microscope.

[0086] Comparative Example 1

[0087] Except for the absence of the glycoprotein denaturation step and the deglycosylation step, the other steps are the same as those in Example 1.

[0088] As can be seen from Example 2, after the colorectal cancer cell line was treated with the deglycosylation enzyme PNGase F, the VISTA protein was clearly detected to be converted from a 40-70 kD band to a bright band of about 36 kD, which improved the detection efficiency of Western blots ( Figure 2A This phenomenon also exists in non-colorectal cancer cells, such as lung cancer and prostate cancer ( Figure 2B ). At the same time, Example 3 can clearly detect that after the colorectal cancer cell line is treated with PNGase F, the fluorescence intensity of VISTA is significantly enhanced ( Figure 2C ). In addition, treatment with glycosylase inhibitors NGI-1 and TM (Tunicamycin) can also significantly enhance the detection efficiency of VISTA in colorectal cancer cells. ( Figure 2D , Figure 2E ).

[0089] Combined with the above experimental results, the applicant found that removing the glycosylation level of VISTA can effectively improve the detection intensity of VISTA in colorectal cancer tumor cells in Western blots and IF. In the subsequent IHC detection process (according to the detection method of Example 1 and Comparative Example 1), the applicant used the deglycosylation enzyme PNGase F to treat the tissue sections, and it was found that the VISTA positive rate of colorectal cancer tumor cells was significantly improved (from 2.63% to 20.5%). At the same time, the VISTA of stromal cells was also improved to a certain extent (from 35.8% to 59.5%) (see Figures 3A to 3E ).

[0090] Effect Example

[0091] 190 intestinal cancer tissue sections were dried, dewaxed, and antigen repaired according to the method steps of Example 1, and then the sections were denatured with 1× glycoprotein denaturation buffer (0.5% SDS, 40mM DTT), and then deglycosylated with 5% PNGase F, followed by inactivation, blocking, and primary antibody application. Corresponding intestinal cancer tissue sections were dried, dewaxed, antigen repaired, inactivated, blocked, and primary antibody applied according to the method steps of Comparative Example 1.

[0092] According to the immunohistochemistry results, the applicant subsequently performed a tissue score (H-score), which is calculated by multiplying the staining intensity (0, negative; 1, weak; 2, moderate; 3, strong) and the percentage of positive staining (the total score range is 0-300). After deglycosylation treatment, the VISTA positive rate was significantly improved, especially the VISTA positive rate of colorectal cancer tumor cells increased from 2.63% to 20.5% ( Figures 3A to 3D ). Moreover, after deglycosylation treatment, the expression of VISTA in colorectal cancer cells is an important marker for poor prognosis of colorectal cancer ( Figure 3E ), which is crucial for the subsequent screening of people who will benefit from VISTA monoclonal antibodies.

Claims

1. A method for detecting VISTA protein in tumor cell lines by immunoblotting, characterized in that The method includes the step of removing the glycosylation modification of VISTA.

2. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 1, characterized in that After the glycoprotein is denatured, a step is performed to remove the glycosylation modification of VISTA.

3. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 1 or 2, characterized in that The tumor cell line is a colorectal cancer tumor cell line, a lung cancer tumor cell line or a prostate cancer tumor cell line.

4. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 2, characterized in that The method comprises the following steps: Step S1, extracting and lysing tumor cells in a tumor cell line to obtain cell proteins; Step S2, denaturing the cell protein into glycoprotein; Step S3, removing the glycosylation modification of VISTA; Step S4, protein concentration determination and glue preparation; Step S5, electrophoresis and membrane transfer; Step S6, first blocking, then incubating with primary antibody and secondary antibody; Step S7: washing and developing.

5. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 4, characterized in that The method comprises the following steps: Step S1, collecting the culture dish where the tumor cell line adherent cells are located, aspirating the supernatant, washing twice with PBS to wash away the residual culture medium, adding about 200 to 500 μL of lysis solution into the culture dish, scraping the cells from the culture dish, transferring them into an EP tube, and ultrasonically lysing them to obtain a cell lysate of cell protein; Step S2, taking the cell lysate, adding 10× glycoprotein denaturation buffer, mixing by pipetting, incubating at 100° C. for 8 to 12 minutes, and then rapidly cooling on ice; Step S3, taking the denatured glycoprotein product, adding sodium phosphate buffer at pH 7.5, NP40 and double distilled water to prepare a reaction system, then adding deglycosylation enzyme, and incubating at 37° C. overnight; Step S4, according to the instructions of the kit, add the protein supernatant obtained by centrifugation and the G250 reagent, gently blow and mix evenly, add the mixed solution to each well of the 96-well plate, and then measure the absorbance at 595nm with an ELISA reader, calculate the protein concentration according to the standard curve, and mark the tube; the gel rapid preparation kit is configured with a separation gel of corresponding concentration; Step S5, fix the gel in the electrophoresis tank, add a sufficient amount of electrophoresis solution, add the protein sample to the corresponding electrophoresis tank, use 80V voltage in the concentrated gel, and use 120V voltage for electrophoresis when the protein and marker are separated and enter the lower separation gel; place the transfer sponge, transfer filter paper, gel, NC membrane, transfer filter paper and transfer sponge in order, clamp them with transfer clips, insert them into the transfer tank, add an ice box, fill with transfer solution, and transfer at 280mA to 320mA for 0.8 to 1.2 hours under constant current conditions; Step S6, soaking the transferred NC membrane in 4-6% skim milk, blocking at room temperature for 0.8-1.2 hours; cutting the target band area from the NC membrane, soaking it in 4-6% skim milk containing VISTA primary antibody at a primary antibody concentration of 1:800-1200, placing it on a shaker, incubating at 4°C overnight or at room temperature for 1.5-2.5 hours; recovering the 4-6% skim milk diluted with the primary antibody, washing the NC membrane with TBST for 2-5 times, each time for 4-8 minutes, and then preparing the secondary antibody corresponding to the primary antibody with TBST, placing it on a shaker, and incubating it at room temperature for 1 hour; Step S7, after the secondary antibody incubation is completed, the NC membrane is washed with TBST for 2 to 5 times, each time for 4 to 8 minutes; the developer is prepared in a volume ratio of 1:0.8 to 1.2, and placed in a luminometer for development.

6. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 5, characterized in that The method comprises the following steps: Step S1, collecting the culture dish where the tumor cell line adherent cells are located, aspirating the supernatant, washing twice with PBS to wash away the residual culture medium, adding about 200 to 500 μL of lysis solution into the culture dish, scraping the cells from the culture dish, transferring them into an EP tube, and ultrasonically lysing them to obtain a cell lysate of cell protein; Step S2, take 18 μL of cell lysate, add 2 μL of 10× glycoprotein denaturation buffer, mix by pipetting, incubate in a metal bath at 100°C for 10 minutes, and then quickly cool on ice; Step S3, take 10 μL of the glycoprotein denatured product, add 2 μL of 500 mM sodium phosphate buffer at pH 7.5, 2 μL of 10% NP40 and 6 μL of double distilled water to make a 20 μL reaction system, then add 1 μL of N-glycosidase F, and incubate at 37°C overnight; Step S4, according to the Bradford kit instructions, add the protein supernatant obtained by centrifugation and G250 reagent at a volume ratio of 10:300, gently blow evenly, add 300 μL of the mixture to each well of the 96-well plate, then measure the absorbance at 595 nm with an ELISA reader, calculate the protein concentration according to the standard curve, and mark the tube; the rapid gel preparation kit is configured with a separation gel of corresponding concentration; Step S5, fix the gel in the electrophoresis tank, add a sufficient amount of electrophoresis solution, add the protein sample to the corresponding electrophoresis tank, use 80V voltage in the concentrated gel, and use 120V voltage for electrophoresis when the protein and marker are separated and enter the lower separation gel; place the transfer sponge, transfer filter paper, gel, NC membrane, transfer filter paper and transfer sponge in order, clamp them with transfer clips, insert them into the transfer tank, add an ice box, fill them with transfer solution, and transfer at 300mA under constant current conditions for 1 hour; Step S6, soak the transferred NC membrane in 5% skim milk and block at room temperature for 1 hour; cut the target band area from the NC membrane, soak it in 5% skim milk containing VISTA primary antibody at a primary antibody concentration of 1:1000, place it on a shaker, and incubate it at 4°C overnight or at room temperature for 2 hours; recover the 5% skim milk diluted with the primary antibody, wash the NC membrane with TBST for 3 times, 5 minutes each time, and then prepare the secondary antibody corresponding to the primary antibody with TBST, place it on a shaker, and incubate it at room temperature for 1 hour; Step S7, after the secondary antibody incubation is completed, the NC membrane is washed 3 times with TBST, each time for 5 minutes; the developer is prepared in a 1:1 volume ratio and placed in a luminometer for development.

7. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 5, characterized in that The protein lysis solution in step S1 is prepared by adding 20 mg / mL PMSF, 100 mmoL sodium fluoride, 200 mmoL sodium vanadate and protease inhibitors to 1% NP40 at a volume ratio of 1:100 and shaking gently to obtain the solution.

8. The method for detecting VISTA protein in a tumor cell line by immunoblotting as claimed in claim 5, characterized in that The glycoprotein denaturation buffer is a 0.5% SDS sodium dodecyl sulfate solution containing 40 mM dithiothreitol.

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